Dataset Information

Identification and preliminary characterization of Hc-clec-160, a novel C-type lectin domain-containing gene of the strongylid nematode Haemonchus contortus.

ABSTRACT:

Background

The strongylid parasite Haemonchus contortus causes severe anemia in domestic animals worldwide. Effective preventive and therapeutical agents are lacking, because of drug resistance and that little is known about the molecular mechanism of the interaction between H. contortus and host cells.

Methods

A new gene, Hc-clec-160, was discovered with RT-PCR. Transcriptional levels of Hc-clec-160 and Ce-clec-160 throughout different growth phases of corresponding nematodes were assayed by qPCR. Immunofluorescence staining of paraffin section were performed to determine the protein localization in adult worms of H. contortus. To monitor the promoter capacity of the 5'-flanking region of Ce-clec-160, micro-injection was used. Overexpression and RNAi constructs was carried out in the N2 strain of Caenorhabditis elegans to find out the gene function of Hc-clec-160.

Results

The full-length cDNA of 1224 bp of Hc-clec-160 was cloned by RT-PCR. The corresponding gene contained twelve exons. Its transcripts peaked in male adult worms. Hc-CLEC-160 was predicted to have a Willebrand factor type A (vWA) domain and a C-type lectin domain. The proteins were not detected by expression in C. elegans or paraffin section experiments in adult of H. contortus. Knockdown of Ce-clec-160 expression in C. elegans by RNAi resulted in shortened body length and decreased brood size.

Conclusions

In this experiment, a new gene Hc-clec-160 was obtained in H. contortus and its function was addressed using a model organism: C. elegans. Our study showed that Hc-clec-160 possesses characteristics similar to those of Ce-clec-160 and plays an important role in the growth and reproduction of this parasitic nematode.

SUBMITTER: Zhang L

PROVIDER: S-EPMC6054721 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe strongylid nematode Haemonchus contortus is a parasite of major concern for modern livestock husbandry because hostile environmental conditions may induce diapause in the early fourth-stage larvae.MethodsA new gene Hc-daf-22 was identified which is the homologue of Ce-daf-22 and human SCPx. Genome walking and RACE were performed to obtain the whole cDNA and genomic sequence of this gene. Using qRT-PCR with all developmental stages as templates to explore the transcription level and micro-injection was applied to confirm the promoter activity of the 5'-flanking region. Overexpression, rescue and RNA interference experiments were performed in N2, daf-22 mutant (ok 693) strains of C. elegans to study the gene function of Hc-daf-22.ResultsThe full length gene of Hc-daf-22 (6,939 bp) contained 16 exons separated by 15 introns, and encoded a cDNA of 1,602 bp (533 amino acids, estimated at about 59.3 kDa) with a peak in L3 and L4 in transcriptional level. The Hc-DAF-22 protein was consisted of a 3-oxoacyl-CoA thiolase domain and a SCP2 domain and evolutionarily conserved. The 1,548 bp fragment upstream of the 5'-flanking region was confirmed to have promoter activity compared with 5'-flanking region of Ce-daf-22. The rescue experiment by micro-injection of daf-22 (ok693) mutant strain showed significant increase in body size and brood size in the rescued worms with significantly reduced or completely absent fat granules confirmed by Oil red O staining, indicating that Hc-daf-22 could partially rescue the function of Ce-daf-22. Furthermore, RNAi with Hc-daf-22 could partially silence the endogenous Ce-daf-22 in N2 worms and mimic the phenotype of daf-22 (ok693) mutants.ConclusionThe gene Hc-daf-22 was isolated and its function identified using C. elegans as a model organism. Our results indicate that Hc-daf-22 shared similar characteristics and function with Ce-daf-22 and may play an important role in peroxisomal β-oxidation and the development in H. contortus.

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